Myoglobin Ligand Research Articles

Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

Myoglobin ligand gate mechanism analysis by a novel 3D visualization technique

A protein is commonly visualized as a discrete piecewise linear curve, conventionally characterized in terms of the extrinsically determined Ramachandran angles. However, in addition to the extrinsic geometry, the protein has also two independent intrinsic geometric structures, determined by the peptide planes and the side chains respectively. Here we develop a novel 3D visualization method that instead of the extrinsic geometry utilizes the intrinsic geometry of side chains. We base our approach on a series of orthonormal coordinate frames along the protein side chains in combination with a mapping of the atoms positions onto a unit sphere, for visualization purposes. We develop our methodology in terms of an example, by analyzing the acidity dependence of the presumed myoglobin ligand gate. In the literature, the ligand gate is often asserted to be highly localized at the distal histidine, its functioning being regulated by environmental changes. Thus, we investigate whether any $${\mathrm{pH}}$$ dependence can be detected in the orientation of the distal and proximal histidine residues, using existing crystallographic data. We observe no $${\mathrm{pH}}$$ dependence, in support of the alternative proposals that the ligand gate is more complex and might even be located elsewhere. Our methodology should help the planning of future myoglobin structure experiments, to identify the ligand gate position and its mechanism. More generally, our methodology is designed to visually depict the spatial orientation of side chain covalent bonds in a protein. As such, it can be eventually advanced into a general visual 3D tool for protein structure analysis for purposes of prediction, validation and refinement. It can serve as a complement to widely used visualization suites such as VMD, Jmol, PyMOL and others.

Renormalization of myoglobin–ligand binding energetics by quantum many-body effects

We carry out a first-principles atomistic study of the electronic mechanisms of ligand binding and discrimination in the myoglobin protein. Electronic correlation effects are taken into account using one of the most advanced methods currently available, namely a linear-scaling density functional theory (DFT) approach wherein the treatment of localized iron 3d electrons is further refined using dynamical mean-field theory. This combination of methods explicitly accounts for dynamical and multireference quantum physics, such as valence and spin fluctuations, of the 3d electrons, while treating a significant proportion of the protein (more than 1,000 atoms) with DFT. The computed electronic structure of the myoglobin complexes and the nature of the Fe-O2 bonding are validated against experimental spectroscopic observables. We elucidate and solve a long-standing problem related to the quantum-mechanical description of the respiration process, namely that DFT calculations predict a strong imbalance between O2 and CO binding, favoring the latter to an unphysically large extent. We show that the explicit inclusion of the many-body effects induced by the Hund's coupling mechanism results in the correct prediction of similar binding energies for oxy- and carbonmonoxymyoglobin.

On the role of thermal backbone fluctuations in myoglobin ligand gate dynamics

We construct an energy function that describes the crystallographic structure of sperm whale myoglobin backbone. As a model in our construction, we use the Protein Data Bank entry 1ABS that has been measured at liquid helium temperature. Consequently, the thermal B-factor fluctuations are very small, which is an advantage in our construction. The energy function that we utilize resembles that of the discrete nonlinear Schrödinger equation. Likewise, ours supports topological solitons as local minimum energy configurations. We describe the 1ABS backbone in terms of topological solitons with a precision that deviates from 1ABS by an average root-mean-square distance, which is less than the experimentally observed Debye-Waller B-factor fluctuation distance. We then subject the topological multi-soliton solution to extensive numerical heating and cooling experiments, over a very wide range of temperatures. We concentrate in particular to temperatures above 300 K and below the Θ-point unfolding temperature, which is around 348 K. We confirm that the behavior of the topological multi-soliton is fully consistent with Anfinsen's thermodynamic principle, up to very high temperatures. We observe that the structure responds to an increase of temperature consistently in a very similar manner. This enables us to characterize the onset of thermally induced conformational changes in terms of three distinct backbone ligand gates. One of the gates is made of the helix F and the helix E. The two other gates are chosen similarly, when open they provide a direct access route for a ligand to reach the heme. We find that out of the three gates we investigate, the one which is formed by helices B and G is the most sensitive to thermally induced conformational changes. Our approach provides a novel perspective to the important problem of ligand entry and exit.

Heme Proteins: The Role of Solvent on the Dynamics of Gates and Portals Revealed by MD Simulations

In the family of respiratory proteins, hemoglobins and myoglobins have been the first to be crystallized in ‘50. Despite the availability of 3D structures, issues regarding the microscopic functioning remain open, such as, for instance, the R to T switching mechanism in hemoglobin or the ligand escape process in myoglobin. Due to the relatively small number of residues, myoglobin is the suitable candidate to investigate the more general structure-function paradigm, being defined as the hydrogen atom of biology. In this work, to complement our recent study on the dynamics of internal cavities of myoglobin[1], the effect of solvation on these intrinsic pathways has been explored. In particular, 60ns-long molecular dynamics simulation of horse heart met-myoglobin was further analyzed and the dynamics of waters residing around/inside the protein with average residence times of up to tens of nanoseconds was monitored. Together with the knowledge obtained previously[1], the analysis of solvent revealed that myoglobin has in fact only few stable hydration sites in which a water molecule can stay for time longer than 2 ns. Strikingly, all of these sites are close to protein/solvent portals observed in previous studies focused on the entry/escape and migration of various ligands in myoglobin[2-4].1. Scorciapino, M. A.; Robertazzi, A.; Casu, M.; Ruggerone, P.; Ceccarelli, M. J. Am. Chem. Soc. 2009, 131, 11825-11832.2. Cohen, J.; Arkhipov, A.; Braun, R.; Schulten, K. Biophys. J. 2006, 91, 1844-1857.3. Ruscio, J. Z.; Kumar, D.; Shukla, M.; Prisant, M. G.; Murali, T. M.; Onufriev, A. V. Proc. Natl. Acad. Sci. USA 2008, 105, 9204-9209.4. Ceccarelli, M.; Anedda, R.; Casu, M.; Ruggerone, P. Proteins 2008, 71, 1231-1236.

Computational Chemistry for Elucidating Protein Function: Energetics and Dynamics of Myoglobin–Ligand Systems

State-of-the-art computational methodologies are used to investigate the energetics and dynamics of photodissociated CO and NO in myoglobin (Mb···CO and Mb···NO). This includes the combination of molecular dynamics, ab initio MD, free energy sampling, and effective dynamics methods to compare the results with studies using X-ray crystallography and ultrafast spectroscopy metho ds. It is shown that modern simulation techniques along with careful description of the intermolecular interactions can give quantitative agreement with experiments on complex molecular systems. Based on this agreement predictions for as yet uncharacterized species can be made.

Fast Time-Resolved Magnetic Optical Rotatory Dispersion Measurements. 2. Confirmation of Mueller Analysis and Application to Myoglobin Ligand Photolysis

Experimental verifications are presented of expressions derived in the preceding paper that describe a simple and sensitive near-null technique for nanosecond time-resolved magnetic optical rotatory dispersion (TRMORD) spectroscopy. Expressions verified include those describing artifacts associated with strain, imperfect Faraday compensation, and photoselection-induced orientation of the sample. The accuracy of MORD data obtained with this method is demonstrated by comparison with MORD curves calculated from the Kramers−Kronig transform of independently measured magnetic circular dichroism (MCD) spectra. The separation of natural and magnetic optical rotations for chiral biological macromolecules and potential artifacts associated with this separation are discussed. This work confirms the prediction in paper 1 in this series that the MORD technique is less sensitive to linear birefringence (arising from strained optics or photoselected samples, for instance) than the related near-null MCD technique previously developed in this lab. Possible problems associated with imperfect Faraday compensation are discussed, as is the prediction in the preceding paper of a coupling between photoselection-induced linear dichroism of the sample and Faraday rotation of the solvent. Photolysis measurements on carbonmonoxymyoglobin demonstrate that an excitation geometry in which the sample is probed along the unique axis of excitation minimizes the latter effect, improving accuracy in MORD measurements on photoselected samples. The TRMORD technique is applied to the ligand rebinding reaction of photolyzed carbonmonoxymyoglobin in Soret-region spectral measurements that demonstrate the ability of this technique to measure multichannel time-resolved magnetooptical data with a precision suitable for global kinetic analysis.

Control and recognition of anionic ligands in myoglobin

Equilibrium and kinetic experiments on site-directed mutants of a synthetic sperm whale myoglobin (Mb) gene have been performed. Results on the reactivity on both ferric and ferrous wild type and mutants Mb's are presented. Analysis of ligand binding to His(E7) Val and His(E7) Val-Thr(E10) Arg mutants compared to wild-type sperm whale, horse and Aplysia limaelna Mb's, shows that the introduction of an arginyl residue at the topological position E10 greatly enhances the stability of the various Mb:heme ligand adducts. Alternative mechanisms of ligand stabilization may therefore be operative in Mb's lacking the distal histidine.